Amyloid  peptide (A), the principal proteinaceous component of amyloid plaques in brains of Alzheimer's disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative ␣-secretase within the A sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPs␣ into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated ␣-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous ␣-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and A fragments confirm the correct ␣-secretase cleavage site and demonstrate a dependence on the substrate's conformation. Our results provide evidence that ADAM 10 has ␣-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer's disease.The amyloid precursor protein (APP) is a type I transmembrane glycoprotein constitutively expressed in many types of mammalian cells. APP is the precursor of the amyloid  peptide (A), the principal proteinaceous component of amyloid plaques in brains of AlzheimerЈs disease patients (1, 2). The A sequence includes 28 amino acids of the extracellular and 12-15 residues of the membrane-spanning region of APP. A is derived by proteolytic processing of the precursor protein from as yet not identified proteases, the -secretase cleaving at the N terminus, and the ␥-secretase cleaving at the C terminus.The major proteolytic pathway of APP is the constitutive secretory pathway that involves cleavage by a putative ␣-secretase within the A sequence at the cell surface (3-5) and in the trans-Golgi network (6-9). Soluble N-terminal APP (APPs␣) fragments of 105-125 kDa (10) are released into the extracellular medium. The membrane-bound 10-kDa C-terminal fragment (p10) produced by ␣-secretase cleavage of APP contains only part of the amyloidogenic A and can be further cleaved by the ␥-secretase to yield a secreted 3-kDa fragment (p3) (11). Because this pathway does not produce intact A, it is nonamyloidogenic and cannot lead to Alzheimer's disease pathology.There has been intensive interest in the secretase enzymes that cleave APP in relation to the pathology of AlzheimerЈs disease. The putative ␣-secretase cleavage site has been precisely determined (12): in human embryonic kidney cells (HEK 293) transfected with various constructs of APP, the soluble form ends at Gln-15 of A, and the N terminus of the cleaved C-terminal fragment begins at Leu-17. It ...
Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to Nasu-Hakola disease, Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), and FTD-like syndrome without bone involvement. TREM2 is an innate immune receptor preferentially expressed by microglia and is involved in inflammation and phagocytosis. Whether and how TREM2 missense mutations affect TREM2 function is unclear. We report that missense mutations associated with FTD and FTD-like syndrome reduce TREM2 maturation, abolish shedding by ADAM proteases, and impair the phagocytic activity of TREM2-expressing cells. As a consequence of reduced shedding, TREM2 is virtually absent in the cerebrospinal fluid (CSF) and plasma of a patient with FTD-like syndrome. A decrease in soluble TREM2 was also observed in the CSF of patients with AD and FTD, further suggesting that reduced TREM2 function may contribute to increased risk for two neurodegenerative disorders.
Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer's disease. In the present report, we identify the ␣-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl--cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted ␣-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of ␣-secretase activity resulted in a decreased secretion of A peptides. Several mechanisms were elucidated as being the basis of enhanced ␣-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl--cyclodextrin; treatment with lovastatin resulted in higher expression of the ␣-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic ␣-secretase pathway and the formation of neuroprotective ␣-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.A myloid- peptides (A), the principal proteinaceous components of amyloid plaques in brains of Alzheimer's disease (AD) patients, are derived from proteolytic cleavage of the amyloid precursor protein (APP), a type I integral membrane protein that is ubiquitously expressed. Both during and after its transport through the secretory pathway to the surface of cultured cells, a fraction of APP molecules undergoes specific endoproteolytic cleavage, most frequently by a scission between amino acids 16 and 17 of the A region (1). This principal secretory cleavage is effected by (a) protease(s) designated as ␣-secretase(s). Soluble N-terminal APP fragments of 105-125 kDa are released into vesicle lumens and from the cell surface; similar species are readily detected in human plasma and cerebrospinal fluid (2). Recently, evidence has been provided that members of the ADAM family (a disintegrin and metalloprotease) act as ␣-secretases (3-5). For ADAM 10, basal and protein kinase C-stimulated ␣-secretase activity and many properties expected for the proteolytic processing of APP have been found (4).The stimulation of ␣-secretase activity and an increase of ␣-secretase cleaved soluble APP (APPs␣) might be beneficial for the treatment of AD for several reasons. In principle, proteolytic cleavage of APP within the A sequence precludes the formation of the amyloid peptides derived from alternative proteolysis of APP with the -secretase cleaving at the N terminus and the ␥-secretase(s) at the C terminus of A peptides (for a recent review of APP processing, see ref. 6). On the other hand, APPs␣ has trophic effects on cerebral neuro...
Summary Beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for β-amyloid (Aβ) production, is elevated in Alzheimer’s disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2α eIF2α-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2α-P phosphatase PP1c, directly increases BACE1 and elevates Aβ production in primary neurons. Preventing eIF2α phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant negative eIF2α kinase PERK, or PERK inhibitor P58IPK blocks the energy deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2α-P, BACE1, Aβ, and amyloid plaques. Importantly, eIF2α-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2α-P, and amyloid load are all correlated in humans with AD. These results strongly suggest that eIF2α phosphorylation increases BACE1 levels and causes Aβ overproduction, which could be an early, initiating molecular mechanism in sporadic AD.
-Secretase (BACE) is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid -peptide. Here, we report that BACE can be phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1. Phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and complex N-glycosylation. Phosphorylation/dephosphorylation affects the subcellular localization of BACE. BACE wild type and an S498D mutant that mimics phosphorylated BACE are predominantly located within juxtanuclear Golgi compartments and endosomes, whereas nonphosphorylatable BACE S498A accumulates in peripheral EEA1-positive endosomes. Antibody uptake assays revealed that reinternalization of BACE from the cell surface is independent of its phosphorylation state. After reinternalization, BACE wild type as well as BACE S498D are efficiently retrieved from early endosomal compartments and further targeted to later endosomal compartments and/or the trans-Golgi network. In contrast, nonphosphorylatable BACE S498A is retained within early endosomes. Our results therefore demonstrate regulated trafficking of BACE within the secretory and endocytic pathway.
Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.
Amyloid -peptide is generated by two sequential proteolytic cleavages mediated by -secretase (BACE) and ␥-secretase. BACE was recently identified as a membrane-associated aspartyl protease. We have now analyzed the maturation and pro-peptide cleavage of BACE. Pulse-chase experiments revealed that BACE is posttranslationally modified during transport to the cell surface, which can be monitored by a significant increase in the molecular mass. The increase in molecular mass is caused by complex N-glycosylation. Treatment with tunicamycin and N-glycosidase F led to a BACE derivative with a molecular weight corresponding to an unmodified version. In contrast, the mature form of BACE was resistant to endoglycosidase H treatment. The cytoplasmic tail of BACE was required for efficient maturation and trafficking through the Golgi; a BACE variant lacking the cytoplasmic tail undergoes inefficient maturation. In contrast a soluble BACE variant that does not contain a membrane anchor matured more rapidly than full-length BACE. Pro-BACE was predominantly located within the endoplasmic reticulum. Propeptide cleavage occurred immediately before full maturation and trafficking through the Golgi.Alzheimer's disease is the most common age-dependent dementia. Pathologically Alzheimer's disease is characterized by the invariant accumulation of senile plaques (1). Senile plaques are predominantly composed of amyloid -peptide (A), 1 which is derived from the -amyloid precursor protein (APP) (2). APP is a type 1 transmembrane protein that maturates during its transport to the cell surface by several post-translational modifications including N-and O-glycosylation (3), phosphorylation (3-5), sulfation (3), and endoproteolysis (2). Upon trafficking to the cell surface, APP can be reinternalized and targeted to endosomes (6, 7). APP can recycle from endosomes back to the cell surface (8) or be targeted to lysosomes (7) for its final degradation (7,9). In polarized cells such as Madin-Darby canine kidney cells and hippocampal neurons, APP is transported to the basolateral compartment (10, 11) and to axons (12).During its transport to the cell surface, APP undergoes endoproteolytic cleavage. ␣-Secretase cleaves APP within its A domain leading to the secretion of APPs-␣ in biological fluids and conditioned medium of cultured cells (3, 13). -Secretase generates the N terminus of the A domain (2), and this cleavage can occur in direct competition to the ␣-secretase cut (14 -16). -Secretase cleavage appears to occur within the Golgi (15, 16) as well as during reinternalization within endosomes (6, 7). The membrane-bound C-terminal fragment generated by the -secretase cut is subsequently cleaved by the ␥-secretase, which results in the physiological generation and secretion of A (2).Interestingly, point mutations associated with familial Alzheimer's disease have been located at all three secretase cleavage sites, and these mutations pathologically affect endoproteolysis of APP (1).Recently major progress has been achieved in...
Alzheimer's disease (AD)-associated ␥-secretase is a presenilin (PS)-dependent proteolytic activity involved in the intramembraneous cleavage of the -amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the -amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent ␥-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific ␥-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid -peptide (A) are generated by an additional cut in the middle of the transmembrane region of CD44. Like A, these CD44 -peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.Intramembraneous proteolysis has been long thought to be an exceptional biochemical pathway involved in the pathological generation of A 1 (1). However, intramembraneous processing of membrane-bound proteins has now been demonstrated to play an important physiological role in regulated nuclear signaling (2). Currently three such proteolytic pathways are known, PS-dependent intramembraneous proteolysis (3), site 2 protease-mediated cleavage (4), and rhomboid-mediated proteolysis (5, 6). The PS-dependent ␥-secretase cleavage is of pivotal importance for A generation (3). A is physiologically produced from the -amyloid precursor protein (APP) through an initial -secretase cleavage followed by the intramembraneous ␥-secretase cut (7). The resulting peptide is secreted and deposited in the AD-defining amyloid plaques. A knockout of the two homologous PS1 and PS2 genes fully abolishes A generation (8, 9). Furthermore, highly specific ␥-secretase inhibitors bind to PSs (10, 11). Mutagenesis of two conserved critical aspartate residues within transmembrane domains (TMD) 6 and 7 of PS1 and PS2 also blocks ␥-secretase function (12, 13), which indicates that PSs may contain an intrinsic aspartyl protease activity responsible for the ␥-secretase cut (14). Indeed, we have identified a novel active site motif in all PSs (15), which is also present in polytopic bacterial aspartyl proteases called type 4 prepilin peptidases (16). Very recently this finding was confirmed by the identification of the same motif in the signal peptide peptidase SPP, another polytopic aspartyl protease (17). However, increasing evidence indicates that ␥-secretase activity resides in a high molecular weight complex (18) composed of PS (19 -21...
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